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The sodium-activated potassium channel Slack (K Na 1.1, Slo2.2, or Kcnt1) is highly expressed in populations of sensory neurons, where it mediates the sodium-activated potassium current (I KNa ) and modulates neuronal activity. Previous studies suggest that Slack is involved in the processing of neuropathic pain. However, mechanisms underlying the regulation of Slack activity in this context are poorly understood. Using whole-cell patch-clamp recordings we found that Slack-mediated I KNa in sensory neurons of mice is reduced after peripheral nerve injury, thereby contributing to neuropathic pain hypersensitivity. Interestingly, Slack is closely associated with ATP-sensitive P2X3 receptors in a population of sensory neurons. In vitro experiments revealed that Slack-mediated I KNa may be bidirectionally modulated in response to P2X3 activation. Moreover, mice lacking Slack show altered nocifensive responses to P2X3 stimulation. Our study identifies P2X3/Slack signaling as a mechanism contributing to hypersensitivity after peripheral nerve injury and proposes a potential novel strategy for treatment of neuropathic pain.

Intake of sugars, especially the fructose component, is strongly associated with the development of obesity and metabolic syndrome, but the relative role of taste versus metabolism in driving preference, intake, and metabolic outcome is not fully understood. We aimed to evaluate the preference for sweet substances and the tendency to develop metabolic syndrome in response to these sugars in mice lacking functional taste signaling [P2X2 (P2X purinoreceptor 2)/P2X3 (P2X purinoreceptor 3) double knockout mice (DKO)] and mice unable to metabolize fructose (fructokinase knockout mice). Of interest, our data indicate that despite their inability to taste sweetness, P2X2/3 DKO mice still prefer caloric sugars (including fructose and glucose) to water in long-term testing, although with diminished preference compared with control mice. Despite reduced intake of caloric sugars by P2X2/3 DKO animals, the DKO mice still show increased levels of the sugar-dependent hormone FGF21 (fibroblast growth factor 21) in plasma and liver. Despite lower sugar intake, taste-blind mice develop severe features of metabolic syndrome due to reduced sensitivity to leptin, reduced ability to mobilize and oxidize fats, and increased hepatic de novo lipogenesis. In contrast to P2X2/3 DKO and wild-type mice, fructokinase knockout mice, which cannot metabolize fructose and are protected against fructose-induced metabolic syndrome, demonstrate reduced preference and intake for all fructose-containing sugars tested but not for glucose or artificial sweeteners. Based on these observations, we conclude that sugar can induce metabolic syndrome in mice independently of its sweet properties. Furthermore, our data demonstrate that the metabolism of fructose is necessary for sugar to drive intake and preference in mice.

Background and Purpose: The P2X3 receptor is an ATP-gated ion channel expressed by sensory afferent neurons and is used as a target to treat chronic sensitisation conditions. The first-in-class, selective P2X3 and P2X2/3 receptor antagonist, the diaminopyrimidine MK-7264 (gefapixant), has progressed to Phase III trials for refractory or unexplained chronic cough. We used patch clamp to elucidate the pharmacology and kinetics of MK-7264 and rat models of hypersensitivity and hyperalgesia to test its efficacy on these conditions., Experimental Approach: Whole-cell patch clamp of 1321N1 cells expressing human P2X3 and P2X2/3 receptors was used to determine mode of MK-7264 action, potency, and kinetics. The analgesic efficacy was assessed using paw withdrawal threshold and limb weight distribution in rat models of inflammatory, osteoarthritic, and neuropathic sensitisation., Key Results: MK-7264 is a reversible allosteric antagonist at human P2X3 and P2X2/3 receptors. Experiments with the slowly desensitising P2X2/3 heteromer revealed concentration- and state-dependency to wash-on, with faster rates and greater inhibition when applied before agonist compared to during agonist application. The wash-on rate (τ value) for MK-7264 at maximal concentrations was much lower when applied before compared to during agonist application. In vivo, MK-7264 displayed efficacy comparable to naproxen in inflammatory and osteoarthritic sensitisation models and gabapentin in neuropathic sensitisation models, increasing paw withdrawal threshold and decreasing weight-bearing discomfort., Conclusions and Implications: MK-7264 is a reversible and selective P2X3 and P2X2/3 antagonist, exerting allosteric antagonism via preferential activity at closed channels. Its efficacy in rat models supports its clinical investigation for chronic sensitisation conditions., (© 2019 The British Pharmacological Society.)

Outer hair cells (OHCs) are highly specialized sensory cells conferring the fine-tuning and high sensitivity of the mammalian cochlea to acoustic stimuli. Here, by genetically manipulating spontaneous Ca 2+ signalling in mice in vivo, through a period of early postnatal development, we find that the refinement of OHC afferent innervation is regulated by complementary spontaneous Ca 2+ signals originating in OHCs and non-sensory cells. OHCs fire spontaneous Ca 2+ action potentials during a narrow period of neonatal development. Simultaneously, waves of Ca 2+ activity in the non-sensory cells of the greater epithelial ridge cause, via ATP-induced activation of P2X 3 receptors, the increase and synchronization of the Ca 2+ activity in nearby OHCs. This synchronization is required for the refinement of their immature afferent innervation. In the absence of connexin channels, Ca 2+ waves are impaired, leading to a reduction in the number of ribbon synapses and afferent fibres on OHCs. We propose that the correct maturation of the afferent connectivity of OHCs requires experience-independent Ca 2+ signals from sensory and non-sensory cells., (© 2019 The Authors. Published under the terms of the CC BY 4.0 license.)

Objectives: To evaluate whether botulinum toxin A (BoNT-A) injection and Lipotoxin (liposomes with 200 U of BoNT-A) instillation target different proteins, including P2X3, synaptic vesicle glycoprotein 2A, and SNAP-25, in the bladder mucosa, leading to different treatment outcomes., Materials and Methods: This was a retrospective study performed in a tertiary teaching hospital. We evaluated the clinical results of 27 OAB patients treated with intravesical BoNT-A injection (n = 16) or Lipotoxin instillation (n = 11). Seven controls were treated with saline. Patients were injected with 100 U of BoNT-A or Lipotoxinin a single intravesical instillation. The patients enrolled in this study all had bladder biopsies performed at baseline and one month after BoNT-A therapy. Treatment outcome was measured by the decreases in urgency and frequency episodes at 1 month. The functional protein expressions in the urothelium were measured at baseline and after 1 month. The Wilcoxon signed-rank test and ordinal logistic regression were used to compare the treatment outcomes., Results: Both BoNT-A injection and Lipotoxin instillation treatments effectively decreased the frequency of urgency episodes in OAB patients. Lipotoxin instillation did not increase post-void residual volume. BoNT-A injection effectively cleaved SNAP-25 (p < 0.01). Liposome encapsulated BoNT-A decreased urothelial P2X3 expression in the five responders (p = 0.04), while SNAP-25 was not significantly cleaved., Conclusions: The results of this study provide a possible mechanism for the therapeutic effects of BoNT-A for the treatment of OAB via different treatment forms. BoNT-A and Lipotoxin treatments effectively decreased the frequency of urgency episodes in patients with OAB.

Hyperalgesia and allodynia are commonly observed in patients with diabetic neuropathy. The mechanisms responsible for neuropathic pain are not well understood. Thus, in this study, we examined the role played by purinergic P2X3 receptors of the midbrain periaqueductal gray (PAG) in modulating diabetes-induced neuropathic pain because this brain region is an important component of the descending inhibitory system to control central pain transmission. Our results showed that mechanical withdrawal thresholds were significantly increased by stimulation of P2X3 receptors in the dorsolateral PAG of rats (n = 12, P < 0.05 vs. vehicle control) using α,β-methylene-ATP (α,β-meATP, a P2X3 receptor agonist). In addition, diabetes was induced by an intraperitoneal injection of streptozotocin (STZ) in rats, and mechanical allodynia was observed 3 weeks after STZ administration. Notably, the excitatory effects of P2X3 stimulation on mechanical withdrawal thresholds were significantly blunted in STZ-induced diabetic rats (n = 12, P < 0.05 vs. control animals) as compared with control rats (n = 12). Furthermore, the protein expression of P2X3 receptors in the plasma membrane of the dorsolateral PAG of STZ-treated rats was significantly decreased (n = 10, P < 0.05 vs. control animals) compared to that in control rats (n = 8), whereas the total expression of P2X3 receptors was not significantly altered. Overall, data of our current study suggest that a decrease in the membrane expression of P2X3 receptors in the PAG of diabetic rats is likely to impair the descending inhibitory system in modulating pain transmission and thereby contributes to the development of mechanical allodynia in diabetes.

Background and Purpose: It is assumed that ATP induces closure of the binding jaw of ligand-gated P2X receptors, which eventually results in the opening of the membrane channel and the flux of cations. Immobilization by cysteine mutagenesis of the binding jaw inhibited ATP-induced current responses, but did not allow discrimination between disturbances of binding, gating, subunit assembly or trafficking to the plasma membrane., Experimental Approach: A molecular model of the pain-relevant human (h)P2X3 receptor was used to identify amino acid pairs, which were located at the lips of the binding jaw and did not participate in agonist binding but strongly approached each other even in the absence of ATP., Key Results: A series of cysteine double mutant hP2X3 receptors, expressed in HEK293 cells or Xenopus laevis oocytes, exhibited depressed current responses to α,β-methylene ATP (α,β-meATP) due to the formation of spontaneous inter-subunit disulfide bonds. Reducing these bonds with dithiothreitol reversed the blockade of the α,β-meATP transmembrane current. Amino-reactive fluorescence labelling of the His-tagged hP2X3 receptor and its mutants expressed in HEK293 or X. laevis oocytes demonstrated the formation of inter-subunit cross links in cysteine double mutants and, in addition, confirmed their correct trimeric assembly and cell surface expression., Conclusions and Implications: In conclusion, spontaneous tightening of the binding jaw of the hP2X3 receptor by inter-subunit cross-linking of cysteine residues substituted at positions not directly involved in agonist binding inhibited agonist-evoked currents without interfering with binding, subunit assembly or trafficking., (© 2014 The British Pharmacological Society.)

Introduction: Cancer pain creates a poor quality of life and decreases survival. The basic neurobiology of cancer pain is poorly understood. Adenosine triphosphate (ATP) and the ATP ionotropic receptor subunits, P2X2 and P2X3, mediate cancer pain in animal models; however, it is unknown whether this mechanism operates in human, and if so, what the relative contribution of P2X2- and P2X3-containing trimeric channels to cancer pain is. Here, we studied head and neck squamous cell carcinoma (HNSCC), which causes the highest level of function-induced pain relative to other types of cancer., Results: We show that the human HNSCC tissues contain significantly increased levels of ATP compared to the matched normal tissues. The high levels of ATP are secreted by the cancer and positively correlate with self-reported function-induced pain in patients. The human HNSCC microenvironment is densely innervated by nerve fibers expressing both P2X2 and P2X3 subunits. In animal models of HNSCC we showed that ATP in the cancer microenvironment likely heightens pain perception through the P2X2/3 trimeric receptors. Nerve growth factor (NGF), another cancer-derived pain mediator found in both human and mouse HNSCC, induces P2X2 and P2X3 hypersensitivity and increases subunit expression in murine trigeminal ganglion (TG) neurons., Conclusions: These data identify a key peripheral mechanism in cancer pain and highlight the clinical potential of specifically targeting nociceptors expressing both P2X2 and P2X3 subunits (e.g., P2X2/3 heterotrimers) to alleviate cancer pain.

P2X receptors are trimeric membrane proteins that function as ion channels gated by extracellular ATP. We have engineered a P2X2 receptor that opens within milliseconds by irradiation at 440 nm, and rapidly closes at 360 nm. This requires bridging receptor subunits via covalent attachment of 4,4'-bis(maleimido)azobenzene to a cysteine residue (P329C) introduced into each second transmembrane domain. The cis-trans isomerization of the azobenzene pushes apart the outer ends of the transmembrane helices and opens the channel in a light-dependent manner. Light-activated channels exhibited similar unitary currents, rectification, calcium permeability, and dye uptake as P2X2 receptors activated by ATP. P2X3 receptors with an equivalent mutation (P320C) were also light sensitive after chemical modification. They showed typical rapid desensitization, and they could coassemble with native P2X2 subunits in pheochromocytoma cells to form light-activated heteromeric P2X2/3 receptors. A similar approach was used to open and close human acid-sensing ion channels (ASICs), which are also trimers but are unrelated in sequence to P2X receptors. The experiments indicate that the opening of the permeation pathway requires similar and substantial movements of the transmembrane helices in both P2X receptors and ASICs, and the method will allow precise optical control of P2X receptors or ASICs in intact tissues.

Background: Electroacupuncture (EA), as a traditional clinical method, is widely accepted in pain clinics, but the analgesic effect of EA has not been fully demonstrated. In the present study, we investigated the effect of EA on chronic pain and expression of P2X3 receptors in the spinal cord of rats with chronic constriction injury (CCI)., Methods: The study was conducted in 2 parts. In part 1, Sprague Dawley rats were divided into 6 groups (n = 10): sham-CCI, CCI, LEA; CCI + 2 Hz EA at acupoints), HEA; CCI + 15 Hz EA at acupoints), NA-LEA (CCI + 2 Hz EA at nonacupoints), and NA-HEA (CCI + 15 Hz EA at nonacupoints). EA treatment was performed once a day on days 4 to 9 after CCI. Nociception was assessed using von Frey filaments and a hotplate apparatus. The protein and the messenger RNA (mRNA) levels of P2X3 receptors in the spinal cord were assayed by Western blotting and real-time polymerase chain reaction, respectively. In part 2, rats were divided into 5 groups (n = 10): sham-CCI, CCI, EA (CCI + EA at acupoints), NA-EA (CCI + EA at nonacupoints), and U0126 (CCI + intrathecal injection of U0126). EA treatment was conducted similar to part 1. Rats were given 5 µg U0126 in the U0126 group and 5% dimethyl sulfoxide intrathecally. Ten microliters was used as a vehicle for the other 4 groups twice a day on days 4 to 9 after CCI. Extracellular signal-regulated kinase 1/2 (ERK1/2) and ERK1/2 phosphorylation in the spinal cord were also assayed by Western blotting., Results: EA treatment exhibited significant antinociceptive effects and reduced the CCI-induced increase of both protein and mRNA expression of P2X3 receptors in the spinal cord. Furthermore, 2 Hz EA had a better analgesic effect than 15 Hz EA, and the protein and mRNA level of P2X3 receptor in spinal cord were lower in rats treated with 2 Hz EA at acupoints than 15 Hz EA at acupoints. Either EA at acupoints or intrathecal injection of U0126 relieved allodynia and hyperalgesia and reduced the expression of P2X3 receptors and ERK1/2 phosphorylation in the spinal cord., Conclusions: The data demonstrated that EA alleviates neuropathic pain behavior, at least in part, by reducing P2X3 receptor expression in spinal cord via the ERK1/2 signaling pathway. Low frequency EA has a better analgesic effect than high frequency HEA on neuropathic pain.

Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder characterized by pain and hypersensitivity in the relative absence of colon inflammation or structural changes. To assess the role of P2X receptors expressed in colorectal dorsal root ganglion (c-DRG) neurons and colon hypersensitivity, we studied excitability and purinergic signaling of retrogradely labeled mouse thoracolumbar (TL) and lumbosacral (LS) c-DRG neurons after intracolonic treatment with saline or zymosan (which reproduces 2 major features of IBS-persistent colorectal hypersensitivity without inflammation) using patch-clamp, immunohistochemical, and RT-PCR techniques. Although whole cell capacitances did not differ between LS and TL c-DRG neurons and were not changed after zymosan treatment, membrane excitability was increased in LS and TL c-DRG neurons from zymosan-treated mice. Purinergic agonist adenosine-5'-triphosphate (ATP) and α,β-methylene ATP [α,β-meATP] produced inward currents in TL c-DRG neurons were predominantly P2X(3)-like fast (∼70% of responsive neurons); P2X(2/3)-like slow currents were more common in LS c-DRG neurons (∼35% of responsive neurons). Transient currents were not produced by either agonist in c-DRG neurons from P2X(3)(-/-) mice. Neither total whole cell Kv current density nor the sustained or transient Kv components was changed in c-DRG neurons after zymosan treatment. The number of cells expressing P2X(3) protein and its mRNA and the kinetic properties of ATP- and α,β-meATP-evoked currents in c-DRG neurons were not changed by zymosan treatment. However, the EC(50) of α,β-meATP for the fast current decreased significantly in TL c-DRG neurons. These findings suggest that colorectal hypersensitivity produced by intracolonic zymosan increases excitability and enhances purinergic signaling in c-DRG neurons.